Germ cells have the unique ability to transmit parental genome information to the offspring. Germline modification has attracted significant attention in the last two decades because it provided a strategy to manipulate genes in vivo, and its application ranged from basic biomedical research to production agriculture (Manipulating the Mouse Embro, (Cold Spring Harbor Press, New York), pp 1-29, 2003). Current techniques to modify germline cells are based on oocytes, eggs or early embryos obtained from females. Developments in egg culture and transfer technology provided the groundwork for the modification of the germline cells obtained from females. However, although the technique is most widely used in mice, attempts to use germ cells obtained from females for other animal species has been limited due to their different reproductive behavior and difficulty in obtaining and manipulating eggs (Reproduction in farm animals, (Lippincott Silliams & Willins, Philadelphia, Pa.), pp 1-40, 2000), and it is difficult to obtain transgenic animals in more than 1% of injected embryos. Thus, there is clearly a need to establish new protocols for germline modification that have wider range of application.
While the female germ cells cease to proliferate before birth, all male germ cells originate from spermatogonial stem cells that have the ability to self-renew themselves (J. Androl., 21, 776-798, 2000). These cells continue to proliferate throughout life and support spermatogenesis. In contrast to differentiated germ cells that have limited life-span, stem cell-based transgenesis has a clear advantage in that transfected stem cells will continuously produce enormous number of transgenic sperm. A single rat stem cell with a transgene can continuously produce ˜2000 transgenic spermatozoa (Histological and Histophathological Evaluation of the Testis, (Cache River Press, Clearwater, Fla.), pp. 1-40, 1990) and therefore numerous transgenic animals can be produced from a founder male. Towards this goal, several groups have succeeded in producing transgenic animals by transducing spermatogonial stem cells in vitro. Spermatogonial stem cells from mice and rats were infected with retrovirus during short-term culture and were transplanted into infertile recipient animals to produce spermatogenesis (Proc. Natl. Acad. Sci. USA, 98, 13090-13095, 2001; Proc. Natl. Acad. Sci. USA, 99, 14931-14936, 2002). By mating with females, recipients produced transgenic animals with efficiency comparable to female-based transgenic methods.
Although this technique provided a new possibility of male germline manipulation, it has several limitations. A major drawback of in vitro transduction approach is the low fertility rate of the recipient animals (Proc. Natl. Acad. Sci. USA, 91, 11298-11302, 1994). One of the reasons is that ablation of endogenous spermatogenesis, which is a prerequisite for efficient colonization of donor cells, often damages the environments of recipient testes for donor cell colonization (Tissue Cell, 31, 461-472, 1999; Biol. Reprod., 69, 412-420, 2003; Dev. Biol., 263, 253-263, 2003; Hum. Reprod., 18, 2660-2667, 2003). Furthermore, the absence of optimal culture condition for stem cells results in the significant decrease in stem cell number (Biol. Reprod., 67, 874-879, 2002; Biol. Reprod., 68, 2207-2214, 2003) and also contributes to lowered fertility. Only 10% of stem cells survive in vitro during 1 week ((Biol. Reprod., 67, 874-879, 2002; Biol. Reprod., 68, 2207-2214, 2003). Due to the rejection of allogeneic donor cells (Biol. Reprod., 68, 167-173, 2003; Reproduction, 126, 765-774, 2003; Biol. Reprod., 69, 1940-1944, 2003), the application of spermatogonial transplantation is still limited in most of other animal species in which immunocompatible recipients are not readily available. Because of these reasons, the efficiency of fertility restoration is limited after spermatogonial transplantation, and prevents the practical application of the technique for transgenesis.
A potentially competitive alternative to produce transgenic animals with spermatogonial stem cells is to introduce genes into stem cells in vivo, because it does not require transplantation or culture of stem cells. However, attempts of such direct transduction of spermatogonial stem cells have met with little success (Mol. Reprod. Dev, 233, 45-49, 1997; J. Virol., 63, 2134-2142, 1989; FEBS Lett., 475, 7-10, 2000; FEBS Lett., 487, 248-251, 2000; Hum. Gene Ther., 9, 1571-1585, 1998; Gene Dev., 1, 366-375, 1987; Biochem. Biophys. Res. Commun., 233, 45-49, 1997). In one study, transgene was integrated in the germline by in vivo electroporation, but the expression did not last long, and disappeared after long-term, indicating that the transgene was introduced into differentiated germ cells (Biol. Reprod., 59, 1439-1444, 1998). In another study, the transgene was not integrated into the germline and was found to be dominantly expressed in Sertoli cells (Biol. Reprod., 67, 712-717, 2002). The difficulty in transfecting spermatogonial stem cells in vivo cannot be explained only by the low number of stem cells in the testis (2 to 3 stem cells per 104 testis cells) (Cell and Molecular Biology of the Testis, (Oxford University Press, New York), pp 266-295, 1993; Mutation Res., 290, 193-200, 1993), since it was not possible with more efficient virus-based approach. Microinjection of various types of virus vectors into seminiferous tubules of adult testes also failed to transduce germline cells in vivo (Proc. Natl. Acad. Sci. USA, 99, 7524-7529, 2002; Proc. Natl. Acad. Sci. USA, 99, 1383-1388, 2002), and no animal studies have shown germline transmission by this approach. It is now considered that stem cells are protected in germline niche, which inhibits the access of the transgenes or virus particles to stem cells (FEBS Lett., 475, 7-10, 2000; Proc. Natl. Acad. Sci. USA, 99, 7524-7529, 2002).
In view of the above-described circumstances, the present invention is intended to provide a method of highly efficiently producing a non-human vertebrate that harbours germ cells having a desired gene transferred thereto by more efficiently transferring a desired gene to male germ cells, particularly spermatogonial stem cells.